The long-term objective of this proposal is to understand the mechanisms by which mediators of the innate immune system induce antigen-nonspecific protection from infectious pathogens and contribute to pathogenesis of disease caused by agents of bioterrorism. Toll-like receptors (TLR) play a critical role in innate immunity by recognizing constitutive and conserved microbial components termed pathogen-associated molecular patterns (PAMPs). Recent evidence suggests that the TLRs can differentially activate innate effector functions, such as type I interferon (IFNalpha/beta), leading to increasing interest in use of synthetic innate immune activators as a novel therapeutic strategy against infection. However, TLRs are also thought contribute to disease pathogenesis of some types of bioterrorism agents by inducing inflammation, immune injury, ultimately resulting in toxic effects on the host. The mechanisms by which TLR-specific signal transduction and gene expression occur are poorly understood. In addition, pathogens may have developed strategies to avoid being eliminated through inhibiting TLR-mediated signal transduction pathways. Over the last three years, we have developed a system to study the signaling, gene expression and functional specificity among the TLRs. Our recent results indicate that certain TLRs (such as TLR9) utilize the p38-dependentsignal transduction pathways to preferentially activate phagocytosis and inflammatory responses. Other TLRs(such as TLR3), use the IRF3 pathway to induce type I interferons and non-specific viral resistance. In the current proposal, we will focus our efforts on understanding the biochemical mechanisms by which the TLRs specifically activate signal transduction pathways to induce innate biodefense. We will determine the mechanisms responsible for TLR-mediated signal specificity at the levels of membrane receptors, cytoplasmic signaling complexes and nuclear transcription factors. Through these studies, we hope to identify potential cellular targets that can separate TLR-mediated antiviral response from TLR-mediated inflammatory response. In order to apply the knowledge we might gain about mechanisms governing TLR-specific immune responses to possible clinical scenarios, we will develop an antiviral model in mice by administering purified or synthetic TLR agonists to determine their effects in the treatment of mice infected with viruses such as influenza. We believe our studies will not only help us to understand the molecular mechanisms responsible for TLR specificity in normal immune responses, but will also provide insightful for design of future therapeutic agents that enhance innate immunity in host biodefense against infectious pathogens.